The transmitter dopamine (DA) is critical for motivation, reward, movement, and cognition. Originating in the ventral tegmental area (VTA), the mesolimbic DA pathway projects to the nucleus accumbens (NAc) core and shell where DA transmission contributes to the rewarding effects of food and drugs, and promotes goal- directed behavior and reward-related learning. Operating in parallel with the mesolimbic system, the nigrostriatal DA pathway originates in the substantia nigra pars compacta (SNc) and projects to the caudate- putamen (CPu). This pathway contributes to motivated behavior and movement, and is involved in driving behaviors that have transitioned from goal-directed and intentional to automatic and compulsive. Given the key roles of these pathways, dysfunction of DA transmission plays a key role in neuropsychiatric disorders including addiction to psychostimulant drugs and the motor deficits of Parkinson's disease. Elucidating causal factors in these disorders, and developing corresponding treatments, requires an understanding of factors that govern DA release. It is often assumed that DA signaling is governed solely by DA neuron firing rate and pattern, with homogeneous activity-dependent changes in extracellular DA concentration ([DA]o) throughout the striatum. However, this is not the case: studies using fast-scan cyclic voltammetry (FCV) show that DA release is regulated locally within the striatum, with temporally and spatially discrete [DA]o transients detected in vivo and in ex vivo brain slices. Preliminary data suggest that a novel source of dynamic local DA release regulation is mediated by glutamate and GABA that are co-released from striatal DA axons. This project will determine autoregulatory roles of co-released glutamate and GABA on axonal DA release in NAc (Aim 1) and CPu (Aim 2), and somatodendritic DA release in VTA and SNc (Aim 3), in ex vivo slices from mice that express channelrhodopsin 2 (ChR2) in DA neurons. Companion studies will assess the presence of glutamate and GABA receptor subunits on striatal DA axons using immuno-electron microscopy. Based on preliminary data showing enhanced AMPA receptor-dependent regulation of striatal DA release in ex vivo slices 24 h after a single cocaine injection, each aim will include examination of the effect of acute cocaine exposure on DA release regulation by co-released transmitters. Overall, this project will define the roles of co-released glutamate and GABA in sculpting how changes in DA neuron activity translate into DA release, and how this regulation can be disrupted by cocaine.